Pig edema disease is a bacterial disease caused by rapid proliferation of Escherichia coli having an Stx2e (edema disease bacterium) in the upper intestinal tract, resulting in absorption of the toxin into blood and is known to occur at a high incidence in baby pigs one or two weeks later of weaning. The fatality of the infection with an edema disease bacterium is very high (50 to 90%). Currently, various antibiotics are used for controlling edema disease, but use of the antibiotics is limited because of problems such as emergence of drug-resistant bacteria.
Because of this situation, studies on pig edema disease vaccines have been made to provide a method of efficiently preventing pig edema disease. For example, a case where pigs are immunized with a detoxified edema disease bacterium-toxin protein to prevent death caused by infection of the edema disease bacterium has been reported (Non-patent Document 1). In this case, a detoxified edema disease bacterium-toxin protein is produced by a recombinant Escherichia coli and inoculated into the pigs. However, this method has a problem of not achieving a low cost from the viewpoint of practical application of a pig edema disease vaccine because an insufficient amount of the detoxified edema disease bacterium-toxin protein produced by the recombinant Escherichia coli and the need of inoculation of the vaccine by direct injection or nasal spray of the protein requires labor of the human.
Meanwhile, studies have been made on production of a useful substance by a plant using a transgenic technology. For example, production of B-subunit of Escherichia coli heat-labile toxin (LT) protein by Lactuca sativa has been reported (Non-patent Document 2). In this study, a B-subunit gene of a codon-modified LT protein is expressed in Lactuca sativa using a cauliflower mosaic virus 35S RNA promoter (CaMV35S) serving as a promoter, which is expressed in a plant at a high level, and Kozak sequence serving as an enhancer. As a result, it has been reported that B-subunit of the LT protein is accumulated in an amount of about 2.0% by mass based on the total soluble proteins of Lactuca sativa. However, the amount of the protein accumulated is considered to be insufficient to efficiently control a bacterial disease using a transgenic plant. That is, it is necessary to efficiently produce and accumulate a target protein in plant cells by expressing the gene of the target protein at a high level.
On the other hand, the base sequence of the 5′-untranslated region (NtADH5′UTR) of an alcohol dehydrogenase gene derived from Nicotiana tabacum is known to be an enhancer capable of acting in Arabidopsis thaliana or Oriza sativa (Patent Documents 1 and 3). However, a case where a gene encoding a bacterial toxin protein is expressed using the base sequence of NtADH5′UTR has not been reported.
[Non-patent Document 1] Makino et al., Microbial Pathogenesis, Volume 31, Number 1, July 2001, pp. 1-8(08)
[Non-patent Document 2] Kim et al., Protein Expression and Purification, Volume 51, Number 1, January 2006, pp. 22-27(06)
[Patent Document 1] JP 2003-79372 A
[Non-patent Document 3] Satoh et al., The 5′-untranslated region of the tobacco alcohol dehydrogenase gene functions as an effective translational enhancer in plant. J. Biosci. Bioeng. (2004)98, 1-8